Decoding butyrate fermentation with glucose as a model substrate: Parameter optimization and metagenomic insights

Selective butyrate production via anaerobic fermentation holds promise for sustainable biochemicals. Despite butyrate frequently accumulate in anaerobic systems, its mechanisms and optimal conditions remain unclear. To address this gap, batch experiments with glucose were conducted to explore the effect of pH (4.5-7.0 and 8.5-11.0), temperature (37 °C and 55 °C) and inoculum-to-substrate ratio (ISR; 3:1, 2:1, 1:1, 1:2, and 1:3) on butyrate production. The results indicated that the optimal conditions were pH 5.5, 37 °C, and an ISR of 1:3, refining previously inconsistent reports. Metagenomic analysis demonstrated that temperature and ISR significantly influenced microbial communities, identifying key butyrate producers: Clostridium, Caproicibacter, Caproicibacterium, Sporolactobacillus, and Ethanoligenens. The functional gene analysis revealed enriched reverse β-oxidation genes under optimal conditions, highlighting the key role of carbon chain elongation in butyrate production. These findings provide mechanistic insights into butyrate biosynthesis and practical strategies for improving selective butyrate production in anaerobic fermentation.